A. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions useful in modulating or inhibiting human telomerase activity. In certain embodiments, the invention concerns the use of these agents in treatment of proliferative cell disorders, particularly for cancers whose proliferation is determined by processive telomerase activity.
B. Description of Related Art
Telomeres play an important role in chromosome organization and stability. Human telomerase is a terminal transferase that adds TTAGGG units onto the telomere end. In general, telomerase activity is not detected in normal somatic cells leading to the implication of telomerase in cancer and the impetus to develop agents that selectively target telomerase activity.
1. Genomic Instability of Cancer Cells
One of the general characteristics of cancer cells is genomic instability. Though it is still unclear what causes this instability, a hypothesis gaining increasing attention is that free chromosome ends, either from chromosome breakage or from loss of the telomere sequences which cap the ends, are prone to illegitimate recombination events. Thus, telomeres provide stability to the chromosomes. However, there appears to be a gradual loss of telomere sequences with each cell division, perhaps because of the end-replication problem. Tumor cells have shortened telomeres, but they also possess greatly elevated levels of the enzyme telomerase to overcome the end-replication problem, while normal cells do not. Thus, telomerase is an attractive target for new anti-cancer agents because of the expected selectivity for neoplastic cells.
2. Telomeres
Telomeres consist of simple DNA repeats at the end of eukaryotic chromosomes and the proteins that bind specifically to those sequences in whole cells (Blackburn, 1991; Zakian, 1989). Telomeric DNA sequences and structures are conserved among widely divergent eukaryotes. The essential telomeric DNA consists of a stretch of a Grich tandemly repeated sequence. Human and other vertebrate telomeres are based on TTAGGG repeat units. The telomere provides a protective "cap" for the end of the chromosome. Broken chromosomes and free DNA ends are susceptible to end-to-end fusion leading to dicentric, ring or other unstable chromosome forms, and to exonucleolytic degradation (Haber, 1984; Mann, 1983; McClintock, 1941; McClintock, 1942; Roth, 1988). By protecting against these events, telomeres prevent loss of genetic information from sub-telomeric regions of the chromosome.
Telomeres, the ends of eukaryotic chromosomes, are composed of tandemly repeated guanine-rich sequences which have an important role in chromosome organization and stability. However, due to the nature of DNA synthesis, the 5' ends of telomeres shorten with each round of replication leaving a 3' overhang that is subject to degradation. This has been described as the "end-replication" problem of linear chromosomes (Watson, 1972; Olovnikov, 1973). The end-replication problem can be overcome by addition of nucleotides to the 3' end of the telomere. A telomere terminal transferase (telomerase) activity was initially discovered in Tetrahymena (Greider & Backburn, 1985). Telomerase activity has since been found in other ciliates (Zahler & Prescott, 1988; Shippen-Lentz & Blackburn, 1989), Xenopus (Mantell & Greider, 1994), yeast (Cohn & Blackburn, 1995), mouse (Prowse et al., 1993), and human cells (Morin, 1989). Telomerase is a ribonucleoprotein in which the internal RNA component serves as a template for directing the appropriate telomeric sequences onto the 3' end of a telomeric primer. The cloning (Greider & Blackburn, 1989) and secondary structure determinations of the Tetrahymena telomerase RNA have determined the template portion of the RNA which has suggested a model for the mechanism of telomerase activity. Telomerase is thought to act by: 1) Telomerase binding to the 3' single-stranded overhang of the telomere (TTAGGG in humans) which base pairs with the complementary bases of the RNA component of telomerase, 2) Nucleotide addition onto the 3' end of the telomere by telomerase using its RNA component as a template, and 3) Dissociation of the newly synthesized telomeric DNA from the RNA template and repositioning to allow for the next round of polymerization. This last step is called the translocation step.
The variety of secondary structures formed by the guanine-rich telomeric sequences involving G-quartets or hairpins(Guschlbauer, 1990; Williamson, 1994) may have an affect on telomerase activity. For example, there is evidence that the G-tetraplex structures formed by telomeric sequences may hinder initial telomerase binding (Zahler et al., 1991). On the other hand, it has been proposed that G-tetraplex formation may actually facilitate the translocation step.
G-quartet structures may also have a role in telomere function. For example, it has been shown that a variety of proteins will preferentially bind to G-quartet structures (Williamson, 1994). Also, the interaction between guanine-rich DNA strands may be involved in the association of chromosomes seen in cells in the presence of varying concentrations of Na.sup.+ (Diaz & Lewis, 1975). The function of chromosomal association is unknown but it has been proposed that it is important in such functions as homologous pairing involved in meiosis (Sen & Gilbert, 1988). Recently, a yeast nuclease (Kem1p) was found to specifically recognize and cut only G-quartet structures (Liu & Gilbert, 1994). Deletion of this enzyme was shown to cause telomere shortening, cellular senescence, and blockage in the pachytene stage of meiosis in yeast (Bahler et al., 1994; Tishkoff et al., 1995; Liu et al., 1995).
Another possible function of telomeres has sparked a great deal of interest in cancer research. It has been recently proposed that telomere length may serve as a "mitotic clock"(Harley, 1995; Shay, 1995). Normal cells in which telomeres shorten to a critical length become senescent (Allsopp et al., 1992; Harley, 1991). In contrast, immortal cancer cells have an unlimited replicative capacity. Due to the findings that telomerase activity is present in a variety of tumor cells, (Chadeneau et al., 1995; Counter et al., 1994; Counter et al., 1995; Kim et al., 1994) it appears that activation of telomerase is one link to cellular immortality. This makes inhibition of telomerase an ideal strategy for anti-cancer therapy. A number of nucleoside reverse transcriptase inhibitors show anti-telomerase activity in human and Tetrahymena (Strahl & Blackburn, 1994; Strahl & Blackburn, 1996).
3. Chromosome End Replication Problem
Telomeres play a critical role in allowing the end of the linear chromosomal DNA to be replicated completely without the loss of terminal bases at the 5'-end of each strand. Watson (1972) and Olovnikov (1971, 1973) independently described the "end-replication" problem, i.e., the inability of DNA polymerase to replicate fully the ends of a linear DNA molecule. All known DNA polymerases require a primer to initiate polymerization that proceeds in 5'.fwdarw.3' direction. After degradation of the RNA primers, filling-in of internal gaps, and ligation events, the parental strand remains incompletely copied. Thus, in the absence of mechanisms to overcome the end-replication problem, the 5' end of the newly synthesized DNA in each duplex is shortened following every round of DNA replication. The 3' single stranded overhang, if not degraded, is converted to a double stranded deletion in the subsequent generation.
4. Telomerase
Immortal cells appear to overcome the end-replication problem by using telomerase to add telomeric DNA repeats to chromosomal ends. Because its mechanism of action involves the copying of an RNA template into DNA, telomerase can be classified as a reverse transcriptase. However, unlike typical reverse transcriptases from retroviruses or lower eukaryotes, it is a ribonucleoprotein that contains its own RNA template as an integral part of the enzyme (Blackburn, 1992). The RNA moiety of telomerase from various ciliates has been cloned and sequenced. For example, the Tetrahymena telomerase RNA moiety is a 159-nucleotide RNA in which a 3'-CAACCCCAA-5' (SEQ ID NO:1) sequence serves as the template for the synthesis of TTGGGG repeats (Greider, 1989). In Euplotes telomerase, a 15 nucleotide portion, 3'-CAAAACCCCAAAACC-5' (SEQ ID NO:2) of a 191 nucleotide RNA was found that could serve as a template for the synthesis of TTTTGGGG repeats (Shippen-Lentz, 1990). It is of interest to note that the Euplotes 191 nucleotide RNA and the Tetrahymena 159 nucleotide RNA share little overall primary sequence similarity. However, despite their divergent primary structures, the Euplotes 191-nucleotide RNA, the telomerase RNAs from T. thermophila, and these of other ciliates can all be folded into similar secondary structures with the putative telomeric template domains for each RNA lying in a corresponding position (Shippen-Lentz, 1989). Studies indicate that human telomerase also contains an endogenous RNA as template (Morin, 1989). The RNA component of human telomerase has been cloned (Feng, 1995).
Telomerase activity from human cells possesses a number of characteristics. First, in the presence of a G-rich human telomeric primer (TTAGGG).sub.3 (SEQ ID NO:3), TTP, dATP and [.alpha.-.sup.32 P]dGTP, a ladder consisting of bands spaced six bases apart will form (Morin, 1989). Second, since telomerase contains an RNA component, telomerase activity can be obliterated in the presence of RNase A. It has been shown that the bands formed in the presence of excess cold nucleotides TTP and dATP and limiting amounts of [.alpha.-.sup.32 P]dGTP (1.56 .mu.M) are indicative of a pause site at the first guanine in the repeating unit of TTAGGG (Morin, 1989).
Evidence to date indicates telomerase is present in tumor cells but not in normal somatic cells. Thus, telomerase is an attractive novel drug target because there is a strong possibility for selectivity. Strahl and Blackburn (1994) has reported that several chain-terminating inhibitors (arabinofuranosyl-guanine triphosphate, Ara-GTP and 2', 3'-dideoxyribofuranosyl guanine triphosphate, ddGTP) efficiently inhibit Tetrahymena and human telomerase (Strahl and Blackburn, 1996).
5. Telomere/Telomerase in Cellular Senescence, Immortalization and Cancer
Early studies on human chromosome ends demonstrated that somatic (peripheral blood) telomeres appeared significantly shorter than germline (sperm) telomeres from the same individual (Cooke, 1986; Allshire, 1988; de Lange, 1990). It is now generally known that in most (if not all) somatic tissues, chromosomes gradually lose their terminal telomere sequence with each cell division (Harley, 1990; Hastie, 1990; Lindsey, 1991; Allsopp, 1992; Shay, 1993; Vaziri, 1993; Klingelhutz, 1994). In contrast, sperm telomeres increase in length with donor age, indicating that telomeres are actively maintained and even elongated in the germline (Allsopp, 1992). One explanation for this difference is that telomerase is active in germline cells but somehow is turned off in normal somatic tissues. This hypothesis has some support as no detectable telomerase activity has been found in extracts of embryonic kidney cells, or normal ovarian epithelium (Counter, 1992; Counter, 1994).
Whether telomere shortening has an impact on the proliferative activity of somatic cells remains unknown. The minimum telomere length required for maintaining full telomere function has not been fully established. Some evidence suggesting that telomere shortening could play a role in cellular aging comes from the analysis of primary human fibroblasts grown in culture (Harley, 1990; Allsopp, 1992). These cells lose approximately 50 bp per doubling and eventually stop dividing at a senescence stage call M1. Cells arrested at the M1 stage can be rescued by a variety of viral agents (Counter, 1992; Ide, 1984; Wright, 1989; Radna, 1989). The virally transformed cells continue to divide for as many as 50 cell divisions before they face another crisis, called M2, which is characterized by a balance of cell divisions and cell death (Counter, 1992). Cells that have bypassed the M1 arrest continue to lose their telomeric DNA, resulting in much shorter telomeres. An average telomere length of approximately 1.5 kbp or less as the cell approaches M2 may not contain sufficient telomere sequence to sustain normal telomere function.
Occasionally, virally transformed cultures yield immortal cells that have overcome the M2 crisis. Interestingly, unlike the senescent cells, the telomere of the immortal cell lines is stabilized by re-activation of the enzyme telomerase. Counter has postulated that telomerase activation is an obligatory step in the immortalization of human cells (Counter, 1992). In support of this hypothesis, telomerase activity was recently detected in metastatic human ovarian carcinoma cells but not in normal control cells, including healthy ovarian epithelium (Counter, 1994).
6 Telomerase Biochemistry
Alternatively, nucleotide analogs that are incorporated into telomeres by the action of telomerase may interfere with the function of the telomeres. For example, some nucleotide analogs so incorporated may block the ability of telomeres to form G-quarted or G-hairpin structures, thereby rendering the telomeres unable to be recognized by protein which specifically bind these structures. In the current model for the mechanism of telomeric DNA synthesis by telomerase (Blackburn, 1990), the 3' nucleotides of the overhang region of the chromosome terminus base-pair with a telomere-complementary sequence in the telomerase RNA. Next, the chromosomal end is extended using the RNA as a template, resulting in the addition of six telomeric nucleotides. Then, the extended DNA terminus unpairs from its RNA template and is repositioned on the 3' portion of the template, becoming available for another round of elongation by telomerase.
One of the most striking features of the telomerase reaction is that it involves not only copying of an internal template, but also an efficient translocation event which occurs after the last [5' most] residue of the template has been copied into DNA. The translocation step has been deduced from the processive nature of the telomerase reaction in a cell-free assay. Thus, telomerase initiates synthesis on a telomeric sequence DNA primer, and in the presence of an excess of the same primer or of a high concentration of a challenging primer, continues to elongate the first primer up to hundreds of nucleotides before dissociation (Blackburn, 1992).
Not all telomerase preparations are processive in the cell-free assay. Nonprocessive telomerase activity has been described in mouse FM3A cells (Prowse, 1993), Tetrahymena (Collins, 1993) and Xenopus (Mantell, 1994). Both processive and nonprocessive telomerases have been identified by the inventors from different cell lines. Intriguingly, the telomerase in S100 extracts of the human HeLa-S3 subline is nonprocessive, while the telomerase in the parental HeLa cells is processive. Whether these are two different enzymes or the same enzyme with different isoforms is currently being investigated. Whether processivity or non-processivity of the activity identified in the biochemical assay is relevant to telomerase function in whole cells remains to be elucidated. Evidence suggesting that telomerase functions nonprocessively in whole cells has been documented (Blackburn, 1992).
7. Deficiencies in the Prior Art
Strahl and Blackburn (1994) have tested AZT-TP, ara-GTP and ddGTP against a non-mammalian Tetrahymena telomerase and human telomerase (Strahl and Blackburn, 1996). Detailed methods and agents for inhibiting telomerase have not been described. In addition, there has been no distinction made between the telomerase produced in some normal cells and the telomerase produced by cancer cell. The identification of therapeutic compounds which have modulation or inhibitory activity against human telomerase is a desirable goal, particularly to identify compounds and develop methods of treatment of cancers in which processive telomerase contributes to the immortality and undesirable proliferation.
Toward this end and because of the important if not entirely understood role of telomerase in cell growth and senescence, there has been an effort to identify compounds that affect telomerase activity. Use of such compounds in controlling cell proliferation has obvious implications in treatment of malignant cancers. A goal of current medical investigation is to understand and treat cellular disorders, preferably to selectively target cancer cells either by altering the telomere, the telomerase, or the enzyme structure and/or by inhibiting telomerase.